AU720285B2 - Process for preparing macrophages, and kits and compositions therefore - Google Patents

Abstract

The invention relates to a process for preparing a composition comprising macrophages, optionally activated, and/or cells derived from monocytes with a strong potential for antigen presentation, said process comprising a stage of culture of monocytes present in the starting composition, this stage being preceding and/or followed by a stage of elimination of all or part of the constituents other than the monocytes present in the starting composition, with the aid of antibodies directed against said constituents, and/or followed by a stage of elutriation. The invention also concerns the compositions of kits for reducing this process to practice.

Description

-1- PROCESS FOR PREPARING MACROPHAGES, AND KITS AND COMPOSITIONS THEREFORE Field Of The Invention The present invention relates to a process for providing a preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response, or precursors thereof. The process is particularly suitable for preparing macrophages and in particular, activated macrophages (also designated as cytotoxic macrophages). There is also provided a system and a kit for use in the provision of the preparation of monocyte derived cells.

Background Of The Invention Macrophages play a major role in the antitumoral response, and they are able to

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be activated by immunological activators against cancer cells (Adams D. And Hamilton Activation of macrophages for tumor cell kill: effector mechanism and regulation. In oo Heppner Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27; Fidler I.: 15 Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. Oeq.

4714, 1985).

"Furthermore, macrophages, or other cells derived from monocytes or from their 4 precursors, with their strong capacity for endocytosis, digestion, and surface antigen presentation, are capable of inducing a specific immune response. In this way, they represent good candidates for the preparation of vaccines, and more specifically cellular autologous vaccines. Macrophages, or other cells derived from monocytes, presenting antigens on their surface specifically capable of inducing a specific immune response, are designated in the text which follows as MD-APCs (Monocyte-Derived Antigen S Presenting Cells).

19901-00 DOC la- The MD-APCs are obtained by culture ofmononucleated cells (monocytes or their precursors), taken from a patient or healthy individual, in the presence of one (or several) antigen(s) which are desired to be expressed as fragments on the surface of the cells obtained at the end of the culture period.

The antigens which may be cultured with the above mentioned mononucleated cells are, for example, antigens expressed by tumor cells (in particular fragments of killed tumor cells) or by pathogens (in particular bacterial or viral protein fragments).

The MD-APCs thus obtained are used for the preparation of vaccines directed against the pathology associated with the antigen co-cultivated with the mononucleated cells (in particular vaccines against tumors or viral diseases) and more particularly for the preparation of autologous cellular vaccines which are advantageously administered to the patient from whom the starting mononucleated cells were taken.

r 0 1901-00.DOC MD-APCs are also able to be obtained by transfection of the above mentioned cells placed in culture, or of differentiated cells, in particular macrophages, obtained after culture, with DNA sequences coding for antigen fragments such as those defined above.

Macrophages presenting cytotoxic activity which is particularly significant (also designated as activated macrophages or MAK (registered trade mark)) as well as a culture medium and process for obtaining said macrophages were the subject matter of international patent application no. PCT/EP93/01232, filed on May 18, 1993.

The process for preparing macrophages defined in the above mentioned international patent application is carried out with a composition rich in blood cells obtained by apheresis from a healthy individual, and includes a stage of monocyte culture in a culture medium containing vitamin D3 1.25-dihydroxyl and GM-CSF (Granulocyte-Macrophage colony stimulating factor).

Advantageously, the monocytes are cultivated in the presence of lymphocytes for about 6 to 7 days, and the differentiated macrophages thus obtained are activated by the addition of y interferon (IFN-y) in the culture medium.

This culture stage is preceded, as in all procedures for obtaining macrophages described up to today, by a stage of separation of, in one part, mononuclear cells, and, in another part, red cells, granulocytes, and part of the platelets contained in the composition derived from blood obtained by apheresis; and by a stage of elimination, by washing part of the blood platelets and anticoagulants remaining after the preceding separation stage.

The above mentioned stage of separation of red cells and granulocytes is generally carried out by centrifugation of the medium containing the monocytes on a density gradient, particularly in a solution having a specific weight of about to about 1.3 g/ml, such as Ficoll Paque solution (Pharmacia) with a specific weight of 1.077 g/ml.

In the above mentioned international patent application, the composition derived from the blood is obtained by apheresis containing approximately 7 to 9x10 9 leukocytes in a volume of approximately 200 ml. After the stage of separation of the red cells and granulocytes by centrifugation on a density gradient, the composition of cellular suspension placed in culture thus contains, in a volume of approximately 500 ml to 1000 ml: Hematocrit (Red cells) 0.1% of the number of the white cells present in the composition Platelets 1010 White cells 3 to 5 x 109 Lymphocytes 50-70% of the number of white cells present in the composition Monocytes 30-50% of the number of white cells present in the composition Polynuclear cells (or granulocytes) 5% of the number of white cells present in the composition The elimination of red cells and granulocytes, which is done beforehand in the culture stage, has always been considered as an indispensable stage, due particularly to the fact that the granulocytes are liable to totally or partially inhibit the differentiation of monocytes into macrophages during the culture stage.

This inhibition would principally be due to the fact that red cells, granulocytes and platelets use the constituents of the culture medium for their own metabolism, thus exhausting the reserves necessary for the development of the monocytes, and leading to acidification of the culture medium in proportions such that the culture stage cannot be completed (usually requiring about 6 to 7 days).

Furthermore, the granulocytes and platelets would be particularly liable to secrete suppresser factors against the functionality of macrophages, such as TGF (transforming growth factor), and prostaglandins.

Therefore, it is currently acknowledged that the granulocytes and as many of the platelets as possible should be separated from the monocytes before being placed in culture with the latter, even though this separation leads to the elimination of about 20% to about 50%, in particular about 30%, of the mononucleated cells present in the medium before separation.

Summary Of The Invention The present invention relates to a new process for obtaining monocyte derived cells such as macrophages or MD-APCsor precursors thereof.

c- I- -3a- In the first aspect of the present invention there is provided a process for providing a preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response, or precursors thereof, comprising: culturing suitable mononuclear cells obtained from blood for a period of time in culture medium; wherein the mononuclear cells are cultured without prior separation of the mononuclear cells from granulocytes and from red blood cells.

In the second aspect there is provided a system when used in a process of the invention for providing a preparation of monocyte derived cells capable of presenting 10 antigen to induce a specific immune response, or precursors thereof, the system comprising: at least one first receptacle containing a starting composition of blood cells for being processed; (ii) at least one second receptacle containing a physiologically acceptable 15 solution for washing cellular constituents of the starting composition; (iii) at least one third receptacle for receiving the physiologically acceptable solution and the starting composition of blood cells from the first and second receptacles, respectively; (iv) at least one fourth receptacle containing culture medium; first transfer means for enabling transfer of the physiologically acceptable solution and the starting composition from the first and second receptacles into the third receptacle to dilute the starting composition of blood cells for said washing; and 19901-00 OC -3b- (vi) second transfer means for enabling transfer of the culture medium from the fourth receptacle to cellular constituents of the starting composition washed with the physiologically acceptable solution and which are to be cultured in the culture medium; wherein the starting composition includes suitable mononuclear cells for being cultured and other said cellular constituents including granulocytes.

There are also provided kits when used to carry out the instant process.

Accordingly, in the third aspect there is provided a kit when used in a process of the invention wherein the kit comprises at least some components of the above described system.

S• 10 The kit may also comprise compositions for use as internal controls for different stages of the process.

Advantageously, the process of the invention may allow greater yields of .iS.

monocyte derived cells or precursors thereof to be obtained compared to conventionally known processes.

15 The present invention will be described hereinafter with reference to a number of preferred embodiments illustrated in the accompanying drawings.

S•9 190 1-00 DOC -4- Brief Description Of The Accompanying Drawings Figure 1 illustrates apparatus for the removal of platelets and washing the product of apheresis, Figure 2 illustrates apparatus for the culture of mononucleated cells for obtaining macrophages or MD-APCs, Figure 3 illustrates an elutriation system arrangement.

Detailed Description Of Preferred Embodiments Of The Invention The present invention relates to a process for obtaining macrophages and MD- APCs, optionally activated, not including a state of separation by physical means, notably by centrifugation on a density gradient in the way described above, in one part, of the red cells and/or granulocytes, and, in another part, of other white cells, notably monocytes, present in the composition derived from blood and obtained by apheresis.

9 The invention particularly relates to any process for preparing a composition containing macrophages or MD-APCs, optionally activated, characterised in that, 15 starting from a composition derived from blood of human origin, rich in blood cells and specifically in white cells such as monocytes, or in precursors of the latter, in particular a composition derived from blood such as that obtained by apheresis, said procedure includes the following stages: advantageously a dilution of said composition derived from blood, particularly in approximately 2 to 3 times the volume of the latter, with the aid of an appropriate physiological solution, a stage of washing said composition derived from blood, advantageously by simple centrifugation, and washing the pellet issued from the said centrifugation, after Srecovery of the pellet in an appropriate physiological washing solution, particularly in a 19901-00 DOC 4a bag (a transfer type bag), effected, for example, by pressure on said bag, thus eliminating the washing solution to another bag or other recipient, to recover a composition depleted of possible anticoagulants, various residues and platelets, optionally, the repetition of the washing stage mentioned above, particularly from 1 to 2 times, a culture stage of the cells contained in the composition derived from blood obtained after the washing stage mentioned above, by placing said cells in an appropriate culture medium, in particular in a bag, preferably a hydrophobic bag, for about 6 to days (particularly for about 6 to 7 days). This culture stage being optionally done in the presence of specific antigens mentioned above, that is, in particular, in the presence of antigens characteristic of tumor cells or of pathogens in the case of MD-APC production, and possibly followed by an activation stage of the macrophages or MD-APCs obtained o in the culture medium, by addition in said culture medium of an activator such as i*.

*o* 19901-0.DOC 'y interferon, said activator being placed in contact with the contents of the culture medium for about 16 to 24 hours, this stage of culture, and optionally activation, being possibly followed by centrifugation of the culture medium and washing of the pellet issued from the centrifugation, in particular following the procedure indicated above, This stage of culture, and optionally activation, may be: Spreceded by a stage of elimination of all or part of the constituents other than the monocytes or their precursors, in particular the platelets, red cells, granulocytes, and lymphocytes, liable to be present in the starting composition, by contacting the composition derived from the blood obtained after the stage of washing preceding the stage of culture, with antibodies directed against all or part of the above mentioned constituents, and recovering the solution containing the monocytes or their precursors, all or part of the above mentioned constituents remaining fixed to the antibodies; and/or followed by a stage of elimination of all or part of the constituents other than the macrophages or MD-APCs by being placed in I contact with the composition derived from blood obtained after the culture stage S: with the antibodies such as described above, and recovery of the solution containing the macrophages or MD-APCs, all or part of the above mentioned S 20 constituents remaining fixed to the antibodies, and/or followed by a purification stage, in particular by elutriation, in which the macrophages or MD-APCs, optionally activated, are separated from the other constituents of the composition obtained after the stage •po of culture, and possibly the stage of activation mentioned above, separated 25 from, in particular, the platelets, red cells and lymphocytes, by physical means.

The invention results from the discovery made by the inventors which shows that, contrary to ideas known in the field, it is possible to bring the culture of monocytes or their precursors into macrophages or MD-APCS to completion with good results when the culture medium contains controlled quantities of red cells, lymphocytes, granulocytes, and platelets.

The invention thus offers the advantage of having access to a process for obtaining macrophages or MD-APCs which enables to get rid of the stage of separation by physical means of, in one part, the mononucleated cells, and more specifically the monocytes, and, in another part, the red cells, granulocytes (and the vast majority if not the totality of platelets) contained in the composition derived from the blood obtained by apheresis, said stage being costly (requiring sophisticated material) and time consuming.

6 Advantageously, in the above mentioned process of the invention, the starting composition derived from the blood of human origin, namely the composition of cellular suspension enriched in blood cells, such as that issued from apheresis and before the stage 6f washing preceding the stage of culture in the procedure described above, is such that it includes a proportion of monocytes greater than about in particular about 10% to 30%, in number of white cells present in said composition.

Such a starting composition is advantageously obtained by the reduction to practice of process of apheresis conducted on a patient, this process being in particular carried out according to any technique and with any material known by any individual skilled in the art, optionally according to a process adapted for obtaining a starting composition such as defined here above and hereafter.

Advantageously, the starting composition derived from blood of human origin mentioned above may contain up to above 3% red cells, and preferably is such that it comprises a proportion of red cells less than about in particular from 0.3% to 0.5 in number of white cells present in said composition.

Advantageously, the original composition derived from blood of human origin mentioned above is such that: the number of platelets is between about 2x10 1 1 to about 6x10 11 20 in a volume of about 150 ml to about 200 ml, the number of white cells is between about 5x10 9 to about 5x10 1 0 in a volume of about 150 to about 200 ml, the proportion of lymphocytes is between about 60% to about 80%, in number of white cells present in the said composition, S 25 the proportion of monocytes is between about 10% to about in number of white cells present in said composition, the proportion of granulocytes is between about 10% to about in number of white cells present in said composition.

The stage of washing the starting composition, preferably repeated 30 2 times, permits the essential elimination of a part of the platelets, and optionally of the reagents used during the process of obtaining the original composition, in particular those of apheresis, such as anticoagulants.

Advantageously, the percentage of platelets eliminated during this stage is about to about The composition obtained after the stage of washing the original composition contains advantageously, in a volume of about 600 ml, the following constituents: Hematocrit: 0.2% to 0.4% in number of white cells present in said composition Platelets: about 2 to about 6 x 1010 -White cells: about 4x10 9 to about 4x10 10 Lymphocytes: about 60 to about 80% in number of white cells present in said composition Monocytes: about 10% to about 20% in number of white cells present in said composition Polynuclear cells: about 10 to about 15% in number of white cells present in said composition.

According to a preferred embodiment of the process of the invention, the stage of culture and differentiation of the monocytes or their precursors into macrophages or MD-APCs is carried out from a composition derived from blood of human origin containing mononucleated cells, namely monocytes or their precursors and lymphocytes, as well as red cells (in appropriate quantities, in particular in the proportions indicated above), granulocytes and platelets, said culture stage being carried out in particular starting from the composition obtained after the stage of washing the starting composition, such as described above.

Concerning the stage of culture itself, it preferably takes place over a period of about 6 to about 10 days in a specific ready to use culture medium based on modified IMDM (Iscove Modified Dubelcco Medium, Gibco), namely the Dubelcco medium modified by Iscove (IMDM) to which is added L- Glutamine (2 mM, Gibco) or advantageously L-Alanyl-L-Glutamine (2 mM, Gibco), pyruvic acid (2 mM, Gibco), Indomethacine (5x10- 6 M, Sigma), mercaptoethanol (3x10- 5 M, Gibco), and non-essential amino acids Gibco). At the time of culture, the medium will be supplemented by 2 to AB+ serum (or by autologous serum) as well as with antibiotics such as penicillin and streptomycin.

The invention more specifically relates to the above mentioned ready to use culture medium, containing L-Alanyl-L-Glutamine instead of L-Glutamine.

The inventors have in fact shown evidence that the above mentioned ready to use culture medium containing L-Alanyl-L-Glutamine can advantageously be conserved at 4 0 C with the group of constituents of the kits of the invention described hereafter, while the above mentioned ready to use culture medium containing L-Glutamine must be conserved at For the differentiation of monocytes into macrophages, the above mentioned culture medium is advantageously supplemented by GMCSF and vitamin D3.

For the differentiation of monocytes or their precursors into MD-APCs, the above mentioned culture medium is used for co-culture with the antigens described above, and is advantageously supplemented by one or many cytokines such as GMCSF, TNF-ct, IL-13, IL-4, and/or by other adjuvants such as calcitriol and histamine.

In the case of obtaining activated macrophages or MD-APCs, an activator, such as y interferon, is advantageously introduced in the above mentioned culture medium about 16 to about 24 hours before the end of the culture.

According to a preferred embodiment of the procedure described above, the composition of the cellular suspension obtained after the culture stage, in a volume of about 600 ml to 2000 ml, is advantageously the following: Lymphocytes: about 2x10 9 to about 2x10 10 Macrophages (or MD-APCs): about 6x10 8 to about 6x10 9 This final composition includes little or no granulocytes, these having for the most part decomposed during the stage of culture.

The yields of the culture stage mentioned above are advantageously about mononucleated cells, and about 50% to about 70% for the differentiation into macrophages or MD-APCs (taking into account the number of starting monocytes).

The above mentioned yields are calculated as the ratio between the number of macrophages or MD-APCs obtained after the culture stage and the number of monocytes present in the composition before culture.

Advantageously, in the preferred embodiment of the process of the invention described above, the above mentioned stage of culture, and optionally activation, is followed by a stage of purification, in particular by elutriation, in which the macrophages or MD-APCs, optionally activated, are separated from the other constituents of the composition obtained after the stage of culture, and optional activation mentioned above, separated in particular from the platelets, red cells and lymphocytes, by physical means.

Advantageously, the stage of culture, and optional activation, carried out in the preferred process mentioned above, is followed by a stage of elutriation represented in figure 3, this being preferably carried out using the following mechanism: a bag (designated B4 in figure 3) containing the composition comprising macrophages or MD-APCs, optionally activated, obtained after the stage of culture, and after optional activation, as well as a bag containing a physiologic solution (also designated as elutriation solution) able to contain the entire elutriation system in a medium viable for the macrophages or MD-APCs, optionally activated, and an air intake are linked to a transfuser with three branches equipped with a filter, once the air intake is closed, a peristaltic pump pulls the contents of the bag B4 into an elutriator equipped with a rotor with an elutriation chamber, the feeding of the entirety of the elutriation circuit with physiologic liquid being assured by the pulling of the contents of the bag containing the elutriation solution, with the above mentioned pump, during the entire duration of the elutriation, the speed of rotation of the elutriation rotor and the delivery from the pump are such that the various constituents of the composition of bag B4, entering the rotor, are separated as a function of their size in the elutriation chamber; the constituents of the smallest size migrate firstly to the top of the chamber (near the exit of the elutriator), while the constituents of the largest size, namely the macrophages or MD-APCs, optionally activated, migrate to the exit of the chamber lastly, the different constituents of the composition in bag B4 thus separated are harvested separately into bags linked to the elutriator; the platelets and various residues of small size are recovered first into bag C1; the red cells are then i recovered into bag C2, then the lymphocytes into bags C3 and C4, and finally 20 the macrophages or MD-APCs into bag El, the different categories of 0:6 constituents mentioned above being extracted from the elutriator as a function of their respective size, either by diminishing the speed of rotation of the elutriator, or by augmenting the delivery of the pump. The recovery of macrophages or MD-APCs into bag El is advantageously able to be carried out 25 by stopping the rotor of the elutriator and opening the air intake; bag El thus contains the composition of macrophages or MD-APCs, optionally activated, advantageously in an essentially pure form.

As a variation of the process described above, the culture stage and differentiation of monocytes or their precursors into macrophages or MD-APCs may be preceded by a stage of elimination of all or part of the constituents other than the mononucleated cells, specifically those other than the monocytes liable to be contained in the composition obtained after the stage of washing the original composition, such as is described above, said elimination stage being not carried out by physical means, but by carrying out immunological reactions between all or part of the above mentioned constituents and antibodies able to recognize said constituents.

The stage of elimination by antibodies mentioned above is advantageously carried out by contacting the composition issued from the stage of washing the starting composition with anti-platelet antibodies, and/or anti-red cell antibodies, and/or anti-granulocyte antibodies, fixed on a solid support, in particular on the walls of a transfer type bag, or on the walls of any other device in which said composition issued from the initial stage of washing is able to be introduced, said device being optionally set up in order to allow the circulation of said composition along the walls on which said antibodies are fixed.

Contacting said composition with said antibodies is carried out either simultaneously with all or part of the antibodies mentioned above, or successively in particular by introduction of said composition into a bag or other device such as is described above containing anti-platelet antibodies, then passing said composition into a second bag containing anti-red cell antibodies, and/or passing said composition into a third bag containing anti-granulocyte antibodies, the lymphocytes being eliminated during the stage of elutriation following the stage of culture.

According to another embodiment the stage of culture, and optional :activation, is: preceded by a stage of contacting the composition issued from the stage of washing the starting composition with the anti-platelet antibodies, and/or anti- S 20 red cell antibodies and/or anti-granulocyte antibodies, fixed on a solid support, in particular in the manner indicated above, contacting said composition with said antibodies being carried out, either simultaneously with all or part of the above mentioned antibodies, or successively in partiular in the manner indicated above, 25 said composition being then transferred into a bag containing the culture medium, and followed by a stage of contacting the composition issued from said stage of culture, and optional activation, with the anti-lymphocyte antibodies, by introduction of said composition into one (or several) bag(s) or other device such as described above, containing anti-lymphocyte antibodies, and recovery the composition of macrophages or MD-APCs, optionally activated, being sought, in an essentially pure form.

Advantageously, when the preferred process above described is carried out by contacting the composition issued from the stage of washing the starting composition in successive contact with anti-red cell antibodies, then anti-platelet antibodies, then anti-granulocyte antibodies, the composition of the cellular suspension thus obtained before the stage of culture, in a volume of about 600 ml to about 2000 ml, is advantageously the following: Red cells: 0.1% Platelets: 1010 White cells: about 3x10 9 to about 3x10 1 0 Lymphocytes: about 60% to about 80% in number of white cells present in said composition Monocytes: about 20% to about 30% in number of white cells present in said composition Polynuclear cells: In the preferred process described above, the composition of the cellular suspension obtained after the culture stage, in a volume of about 600 ml to 2000 ml, is advantageously identical to that of the cellular suspension obtained after the culture stage in the preceding preferred procedure, not requiring a stage of immunological reaction previous to the culture stage, namely, Lymphocytes: about 2x10 9 to about 2x10 10 Macrophages: about 6x10 8 to about 6x10 9 The yields of the culture stage mentioned above are advantageously identical to those described in the preceding preferred procedure, namely about mononucleated cells, and about 50% to about 70% for the differentiation into macrophages (taking into account the starting number of monocytes).

The yields of the processes described above are advantageously of the same order, be these processes carried out according to the principle of elutriation after the culture stage, or according to the principle of the immunological reaction before and after the culture stage.

Thus, the above described processes of the invention are such that one can obtain cellular suspensions comprising macrophages or MD-APCs, optionally activated, in an essentially pure form, in particular in proportions of about to about 95%, and with final yields of about 20% to about 50% (said yields being calculated as a ratio between the quantity of purified macrophages or MD- APCs obtained at the end and the quantity of monocytes initially placed in culture).

According to a specifically advantageous embodiment of the processes of the invention described above, the composition of the starting cellular suspension, and/or at least one of the compositions of cellular suspension obtained under the different stages of the above mentioned procedures, and/or the composition of the final cellular suspension, is/are able to be compared to the compositions called internal controls, in order to make a good follow up of the processes of the invention.

An internal control particularly advantageous is that which is constituted of a determined quantity of monocytes, purified from blood taken from a healthy individual, advantageously fixed, in particular by lyophilization, said internal control allowing for the quantification of the number of monocytes present in the starting composition.

An internal control specifically preferred is that which includes about 2x10 6 to about 4x10 6 lyophilized monocytes in a volume of about 1 ml to about 2 ml of an aqueous solution.

The quantification of the number of monocytes present in the original composition is advantageously carried out by withdrawing an aliquot part of the starting composition, labelling the monocytes liable to be present in the fraction withdrawn, in particular with the aid of labelled antibodies, for example by radioactivity, enzymes, or by fluoresceine, revealing the labelling carried out by using any appropriate device, and comparing the intensity of the labelling obtained under the same operating conditions with said internal control.

The labelled antibodies able to recognize specific surface antigens of monocytes are advantageously those directed against, for example, the following antigens: CD14, HLADR, and CD64.

Another internal control specifically advantageous is that constituted from a determined quantity of macrophages or purified MD-APCs, optionally activated, from blood withdrawn from a healthy individual, advantageously fixed, in particular by lyophilization, then reconstituted in an adequate solution of 1 to 2 ml, said internal control allowing for the quantification of the number of macrophages or MD-APCs, optionally activated, present in the final composition.

An internal control specifically preferred is such that it includes about 2x10 6 to about 4x10 6 lyophilized macrophages in a volume of about 1 ml to about 2 ml of an aqueous solution.

The quantification of the number of macrophages present in the original composition is advantageously carried out by withdrawing an aliquot part of the original composition, labelling the monocytes present in the fraction withdrawn, in particular with the aid of labelled antibodies, for example by radioactivity, enzymes, or by fluoresceine, revealing the effected labelling by using any appropriate device, and comparing the intensity of the labelling obtained under the same operating conditions with said internal control.

The labelled antibodies able to recognize specific surface antigens of macrophages and/or MD-APCs are advantageously directed against at least one of the following antigens: CD54, CD58, CD80, CD86, CD14, CD16, MAX-1, 13 CD64, HLADR described in particular in the following articles: Andreesen et al., Biology 47: 490-497 (1990), Lopez et al., Journal of Immunotherapy 11: 209-217 (1992), Chokri et al., Anticancer Research 12: 2257-2260 (1992).

Advantageously, the internal controls mentioned above are constituted from beads, chosen in particular from among fluorescent beads, having a diameter comparable to that of monocytes or macrophages, and able to give signals identical to those given by monocytes and macrophages in, respectively, the starting and final solutions.

According to another specifically advantageous embodiment of the processes of the invention described above, the control of the compositions of macrophages or MD-APCs, optionally activated, obtained when reducing to practice these processes, can be carried out by detection, and optionally measure of cytokines secreted by the macrophages in said compositions, and compared to the standard secretion profile of cytokines by macrophages or MD-APCs, optionally activated.

As an illustration, the detection of secreted cytokines such as IL-1 (Interleukin IL-6 and TNF-ca (Tumor Necrosis Factor-ct) can be carried out by withdrawing an aliquot part of culture medium in which the differentiation and the activation of macrophages obtained has taken place.

20 The macrophages and MD-APCs, optionally activated, obtained by the process of the invention, may be tested for their capacity to recognize and kill tumor cells (in particular by cytotoxicity test) or, in the case of MD-APCs, for their capacity for antigenic presentation, in particular by verifying their capacity to induce allogenic lymphoproliferation (for example the MLR test, Mixed 25 Lymphocyte Reaction). These tests allow to determine the number of functional :units of macrophages or MD-APCs obtained during the process of the g: invention.

The invention also relates to kits (or cases) for obtaining compositions of macrophages or MD-APCs, optionally activated, characterized in that they may comprise: the necessary elements for washing, culture, and elutriation, including: one or several washing bag(s), one or several culture bag(s), preferably hydrophobic, one or several connector(s) and needle(s) for the elimination of the supernatant of the washings, one or several connection tube(s) between the preceding bags, optionally, one or several injection site(s) in the washing and culture bag(s), one or several systems allowing for the opening or the closing of the links between the washing bag(s) and the culture bag(s), in particular, systems of clamps on the tubes, two bags containing the physiologic solution for the washing and the elutriation, a three-branch transfuser with air intake, a silicon tube and a joint on the rotor serving as a connection between the transfuser and the rotor of the elutriator, one (or several) collection bag(s), optionally linked together by tubes equipped with a clamp system, a tube and joint on the rotor serving as a connection between the collection bag(s) mentioned above and the rotor of the elutriator, the reactive agents necessary for the culture, differentiation, activation, and control, including: a bag of specific, ready to use culture medium, in particular a culture medium primarily constituted from modified IMDM, namely Iscove modified Dubelcco culture medium (IMDM, Gibco), to which has been added L-Glutamine (2 mM, Gibco) or L-Alanyl-L-Glutamine (2 mM, Gibco), pyruvic acid (2 mM, Gibco), Indomethacine (5x10- 6 M, Sigma), mercaptoethanol (3x10- 5 M, Gibco), non-essential amino acids Gibco), and 2 to 5% of AB+ serum (or autologous serum), as well as antibiotics such as penicillin and streptomycin, added at the moment of culture, one or several supplements advantageously under liquid or lyophilized form, to be added to the culture medium, chosen from among the following:

GMCSF,

vitamin D3, interleukin-13 (IL-13) IL-4 TNF-a histamine optionally, an activator such as y interferon, advantageously under liquid or lyophilized form, advantageously one (or several) internal control(s) comprising monocytes, and/or macrophages or MD-APCs, the latter being optionally activated, advantageously fixed, or lyophilized, and optionally labelled, or calibrated beads, optionally labelled, in particular by fluorescence, one or several labelled antibodies, in particular by fluorescence, such as those directed against antigens CD3 (marker for lymphocytes used in case of negative control), CD45 (marker for leukocytes in general), CD14, CD16, CD64, CD54, CD58, CD80, CD86, HLA-DR and MAX-i.

A kit specifically advantageous in the framework of the present invention is presented in the form of two groups: group I contains the bags, tubes, injection sites, clamp systems, and three-branch transfuser, namely, the "dry" elements used for washing, culture, and the purification by elutriation, group II contains a bag containing the specific culture medium as well as the reactive agents for the culture, differentiation, activation, and quality control.

The kit mentioned above is moreover characterized in that: group I is constituted of two sub-groups in the form of two boxes: box A containing the material necessary for washing the cells obtained from apheresis, in particular: three bags with a capacity of 600 ml (central bag Al, and recovery bags A2 and A3 shown in figure 1), a transfer set for transferring the washing solution into the bag (bag Al in figure 1) in which is transferred the blood composition in order to dilute and wash the cells issued from apheresis, a transfer set for the washed cells (contained in bag Al in figure 1) to the culture bags (bags B1, B2, and B3 in figure 2), a transfer set for the culture medium, firstly towards the washed cells (contained in bag Al in figure and secondly towards the culture bags mentioned above, three hydrophobic culture bags with a capacity of 3 litres (bags B1, B2, and B3 in figure 2), box B contains the elements for the separation of the differentiated macrophages or MD-APCs from the other cells present in the culture during the stage of elutriation, in particular: a gathering bag- (or pooling bag B4 in figure 2), a three-branch transfuser with air intake, a silicon tube, and two joints to the rotor, the tube and one of the two joints serving as a connection between the transfuser and the rotor of the elutriator, the other joint serving as a connection with said rotor and the group of bags mentioned hereafter, the latter tube containing a site for sample withdrawal, a combination of four recovery bags (bags Cl, C2, C3, and C4 in figure 3) with a capacity of one litre, a bag (bag El in figure 3) with a capacity of 600 ml to contain the final fraction of purified macrophages or MD- APCs, and a bag (bag E2 in figure 3) with a capacity of 600 ml for the recovery of the supernatant of the preceding bag, and a bag (bag E3 in figure 3) for injecting the patients with macrophages or MD-APCs, optionally activated, group II is represented by box C, an isothermal box, optionally with a cooling system to maintain the temperature at 4 0 C, and containing in particular: a bag of 2 litres of culture medium primarily constituted from modified IMDM, as described above, a box containing the following liquid or lyophilized products: a flask of antibiotics (penicillin, advantageously 1,000 U/streptomycin, advantageously 1,000 pig), a flask of vitamin D3 (advantageously 4 to 8 pg), a flask of GMCSF (advantageously 5 x 105 to 106 U), a flask of y interferon (advantageously 25 x 104 to 5 x 105

U),

a flask of IL-13, a flask of IL-4, a flask of TNFoa, a flask of histamine, a box containing one (or several) internal control(s) as described above, and one or many flasks containing the labelled antibodies anti- CD3, anti-CD45, anti-CD14, anti-CD16, anti-CD64, anti-MAX-1, anti-CD54, anti-CD58, anti-CD80, anti-CD86, and anti-HLA-DR, in particular labelled with fluoresceine.

The invention also refers to the use of a kit as defined above for the realization of a process as described above according to the invention including a stage of elutriation.

The invention also relates to a kit for obtaining compositions of macrophages or MD-APCs, optionally activated, characterized in that it includes: one (or several) bag(s) inside of which fixed anti-red cell antibodies are found, one (or several) bag(s) inside of which fixed anti-platelet antibodies are found, one (or several) bag(s) inside of which fixed anti-granulocyte antibodies are found, one (or several) bag(s) inside of which fixed anti-lymphocyte antibodies are found, one (or several) culture bag(s), one (or several) bag(s) for the recovery of purified macrophages or MD-APCs, optionally activated, linking tubes between the different bags mentioned above, supplements advantageously under liquid or lyophilized form, to be added to the culture medium, in particular: antibiotics such as penicillin and streptomycin,

GMCSF,

vitamin D3, I* IL-13 IL-4 TNFa histamine optionally, an activator such as y interferon, advantageously under liquid or lyophilized form, advantageously one (or several) internal control(s) comprising 25 monocytes, or macrophages or MD-APCs, optionally activated, advantageously Sfixed, and optionally labelled, or of calibrated beads, optionally labelled, in particular by fluorescence, such as is described above, labelled antibodies, in particular by fluorescence, such as those directed against the antigens CD3, CD45, CD14, CD16, CD64, CD54, CD58, CD86, MAX-1 and HLA-DR.

The invention relates more specifically to the use of a kit as described above for carrying out a process as described above according to the invention comprising a stage of immunological reaction.

A composition of blood cells for use in a process of the invention may comprise a cellular suspension derived from blood of human origin, characterised in that it includes a proportion of monocytes greater than about in particular about 10% to about 30%, in number of white cells present in said composition.

18 A composition of blood cells for use in the process may include a preparation of red cells less than about 3% or less than about in particular from 0.3% to in a number of white cells present in said composition.

Preferably, the composition is characterised in that: the number of platelets is between about 2x10 1 1 to about 6x10 1 1 in a volume of about 150 ml to about 200 ml, the number of white cells is between about 5x10 9 to about 5x10 10 in a volume of about 150 ml to about 200 ml, the proportion of lymphocytes is between about 60% to about in number of white cells present in the said composition, the proportion of granulocytes is between about 10% to about in number of white cells present in said composition.

The invention refers more specifically to the above mentioned compositions such as obtained in the process of apheresis, carried out in .particular on a volume of blood of approximately 5 to 10 litres.

The invention also relates to the use of a composition such as described above for obtaining a composition of macrophages or MD-APCs, optionally S 20 activated, in particular by carrying out a process according to the invention, said composition being able to be administered to a patient, optionally after dilution of the macrophages or MD-APCs, optionally activated, thus obtained in an appropriate injectable solution.

The invention also relates to the compositions (in particular 25 pharmaceuticals or vaccines) containing macrophages and/or MD-APCs, optionally activated, in particularly in the proportions of purity indicated above, and such as are obtained by carrying out the process of the invention, optionally in association with an acceptable pharmaceutical vehicle.

1 Compositions can be used as internal controls in the framework of reducing to practice a process according to the invention, such as described above.

As an example, the internal control in the framework of the preparation of activated macrophages is presented in the following manner: Contents: 1 flask of lyophilisate (3xl0 6 /flask) 1 flask of reconstitution solution.

Reconstitution: Rehydrate the flask of lyophilisate with 1 ml of the solution of reconstitution solution.

Mix gently by turning. Wait at least 10 minutes before using the preparation.

Use: the volume of internal control used is 100 /l (3x10 5 cells) add the antibody to be tested incubate for 30 minutes at room temperature add 1 ml of PSB (do not wash) analyze by cytofluorometry The percentage and intensity of labelling of prepared macrophages or MD-APCs will be compared to the cells produced.

Table 1 which follows gives an indication of the evolution of membrane markers in relation to time of storage, of the lyophilized macrophages used as internal controls. The results are expressed as percentage of positive cells and intensity of labelling. The line "Ctrl" corresponds to the results obtained with fresh, non-lyophilizod macrophages, cultivated and obtained according to the procedure of the invention. The line "7 Days" corresponds to macrophages obtained according to the process of the invention, lyophilized and rehydrated 7 days after lyophilization. The line "7 months" corresponds to macrophages obtained according to the process of the invention, lyophilized and rehydrated 7 months after lyophilization. An overall statement can be made that the percentages of positive cells as well as the intensities of labelling are kept equivalent to or similar to the fresh macrophages and those which are conserved in a lyophilized state for 7 days or 7 months. Thus, the user of the kits of the invention is able to compare the compositions which will be obtained with the internal controls (or standards) furnished with the kit, said controls being advantageously produced according to the same process and being lyophilized.

Table 1 Markers CD3 CD14 CD16 CD64 HLADR Ctrl 92/115 0.3/54 87/149 22/35 77/63 84/115 7 Days 95/131 3/30 96/119 10/30 93/59 96/169 7 Months 91/119 3/22 86/95 10/25 81/54 82/116 Advantageously, the kits of the invention are accompanied by written procedure forms for the preparation of macrophages or MD-APCs, optionally activated.

Advantageously, the kits of the invention are also accompanied by automated computerized instructions to guide the user through the processes and validate each step of the processes of the invention, the final goal being to determine the above mentioned number of functional units obtained by carrying out the processes of the invention.

As an illustration, the written procedure forms for the step of activating macrophages, accompanying a kit according to the invention, can be presented in the following manner: ACTIVATION OF MACROPHIAGES (continued 1) Signature of operator SRemove bag BI from the incubator and complete steps through under a flux laminar hood: Brand: Model no.: Withdraw volume Vi of gamma interferon solution using a sterile, 1 ml syringe.

Withdrawal completed: YES

NO-

Inoculate the solution of gamma interferon into the bag.

Without removing the syringe, aspirate and reinject its contents until homogenization.

Withdraw from the bag under sterile conditions and in one single withdrawal 1 ml to be distributed into two tubes as follows: 0.5 ml of cellular suspension for cell count, viability, and size control (see appendix No.7 page 33).

0.5 ml of cellular suspension for bacterial control (see appendix No.7 page Remove bag B2 from the incubator and repeat steps through Add ml to each of the two tubes already used for the controls for bag B 1.

SRemove bag B3 from the incubator and repeat steps through Add ml to each of the two tubes already used for the controls for bags BI and B2.

Samples taken: Bag 1: YES Bag 2: YES Bag 3: YES NOD NOD NOD Signature of person responsible for this preparation: MVAW® PREPARATION a Lit i cr n Co d e: II) aI c': ACTIVATION 01F MACROPII1AGES I cunui'.-.:ed 2) -Signat Z LU re Altach bar code labels to the two tub~es containing, the withdrawls.

"l Bacteriological Control Day 6 Dlstina FiU in the form "Bacteriological Control Day 6" whichi is located in the MAK Liaison Documnents folder, and send the form with the withdrawls to the destination written above.

o Circle the word "ACTIVIATION" on bags BI, B2 and o Put bags B1, 132 and B3 back in their initial location in the incubator (refer to page 9).

o Destroy any remaining gamma interferon.

Date of destruction: Signature: 0 Complete below: PREPARATION OF HUMAN AUTOLOGOUS

MAK

ATTACH ACTIVAION BAR CODE LfBEL 1-1ER E Da te: Signature of person responsible for this preparation: aae ~agc Description of the figures: The description of the operations represented by figures I through 3 is as follows: I-REMOVAL OF PLATELET FROM THE PRODUCT OF APHERESIS (figure 1) Transfer the contents of the apheresis bag into bag Al (600 ml capacity).

To the bag containing the mononucleated cells, add a weight of buffered saline solution such that the final weight of the bag is 450-500 g.

Centrifuge bag Al at 300 g for 10 minutes at +4 0

C.

Transfer the supernatant into bag A2, using a press.

Fill to 450 g with saline solution.

Centrifuge bag Al a second time.

Transfer the supernatant into bag A3.

Detach the cells from the centrifugation bag Al by friction.

Fill to 600 ml with culture medium which has been previously prepared.

Note the volume of culture medium added.

Withdraw the control samples under sterile conditions.

II-CULTURE OF MONONUCLEATED CELLS (figure 2) Distribute equally into the culture bags Bl, B2 and B3.

Fill the volume required for each bag with culture medium.

(11) At the end of the culture and activation periods, transfer the contents of the three culture bags into the pooling bag B4.

III-SEPARATION BY ELUTRIATION (figure 3) Attach part I (entry of Kit) to the entry of the sterilized elutriation chamber and the exit of the kit (part II) to the exit of the elutriation chamber (12) Fill the circuit with at least 300 ml of elutriation solution.

(13) Simultaneously, close the entryway of the elutriation solution and open that of the cellular suspension bag (bag B4) without changing the delivery of the pump.

As soon as all of the cells have passed, close the entryway of the bag B4 of cells and open that of the elutriation solution.

Increase the delivery of the pump every 500 ml and withdraw a sample.

(14) When cells having the size of macrophages begin to exit, stop the centrifugation and deviated the withdrawal to the collection bag El by closing the entryway of the elutriation solution and opening the air intake.

-24- Centrifuge bag El at 400 g for 10 min.

(16) Transfer the supernatant into bag E2.

(17) Put the cells back into suspension in a solution of 250 ml of human albumin at Homogenize well.

(18) Transfer into the injection bag E3.

The legend of figures 1 to 3 is the following: represents the sense of fluid circulation represents the soldered joint -C represents the perforators 10 represents the clamp represents the connection **Ir represents the connection site Example 1 comparison was made between the yield of activated macrophages following culture of mononuclear cells obtained by difference processes.

1.1 Mononuclear cells A concentrated preparation of leukocytes was obtained by apheresis using a COBE Spectra continuous flow separator. Cell preparation and culture was preformed under different conditions. N represents the number of experiments performed.

Condition 1 (purified monocytes): Leukocytes from apheresis residue were centrifuged over ficoll to remove granulocytes and contaminating red cells. A monocyte population was purified using a elutriation technique and then was seeded in culture as described.

19901-00 DOC 24a Condition 2 (monocytes lymphocytes).

Leukocytes from apheresis residue were centrifuged over ficoll and washed three times in phosphate buffered saline without calcium and magnesium. The mononuclear cells were put in culture as described.

Condition 3 Leukocytes from apheresis residue were washed three times in phosphate buffered saline without calcium and magnesium to remove platelets. Cell product containing polynuclear and red cells was seeded in culture as described.

1.2 Culture and differentiation S 10 Differentiated macrophages were obtained by 7 days culture of mononuclear .o cells.

The culture medium comprised Iscove's modified Dubelco medium, containing L-Glutamine (2mM), Pyruvic acid (2mM), Indomethacin (5 x 10-6 Mercaptoethanol (3 x 10 M) and non-essential amino acids Endotoxin free buffers were used for I5 elutriation (where applicable) and for washing.

Total leukocytes (condition mononuclear cells (condition 2) or purified **.monocytes (condition 1) were seeded at 5 x 106 cell/ml during 7 days in air permeable hydrophobic culture bags at 370 C, 5% CO2, and 95% humidified atmosphere. The specific medium was supplemented with GM-CSF (500 U/ml, Novartis) and 2% of AB or autologous serum. Activation of macrophages to cytotoxicity was performed by a further 16 hours culture with 250 U/ml of IFNy (Boehringer Ingelheim, France).

The yield of MAK recovered after 7 days culture was based on the number of differentiated macrophages obtained at day 7 compared to the number of monocytes in Sculture at day 0.

19 01 -00 DOC 24b The results are shown in Table 2 below.

CELLULAR STARTING MATERIAL CONDITION 1 CONDITION 2 CONDITION 3 Composition Purified Monocytes Lymphocytes Containing Cells N=3 Monocytes Suitable For N=6 Culture Yield Of Human Macrophages After 30 40 50 60 70 90 7 Days Culture The yield of differentiated macrophages using the process of present invention (condition 3) is 2 fold higher than using purified monocytes (condition 1) as the starting cellular material. The culture of selected monocytes lymphocytes (condition 2) gives 20 less recovery of macrophages compared to the process of the present invention.

Although the present invention has been described hereinbefore with reference to a number of preferred embodiments, the skilled addressee will appreciate that numerous variations and modifications are possible without departing from the scope of the 10 invention.

1990 .00. DOC

Claims (17)

1. 2. A process according to claim I further comprising: providing a starting composition of blood cells derived from said blood; and washing said starting composition with a physiologically acceptable 15 solution to obtain a composition containing the mononuclear cells for being cultured.
2. 3. A process according to claim 2 wherein the mononuclear cells are cultured in the presence of platelets which have not been eliminated during the washing step.
3. 4. A process according to claim 2 or 3 wherein said starting composition contains from between about 2 x 10 to about 6 x 10 platelets per 150 ml to 200 ml of the composition. A process according to any one of claims 2 to 4 wherein total white blood cell said cellular constituents present in said starting composition include from about 60% to about 80% lymphocytes in number. 1990I-ODOC -26-
4. 6. A process according to claim 5 wherein the white blood cells in the starting composition include from about 5% to about 30% monocytes in number.
5. 7. A process according to claim 5 or 6 wherein the white blood cells in the starting composition include from about 10% to about 20% granulocytes in number.
6. 8. A process according to any one of claims 5 to 7 wherein said starting composition contains from about 5 x 10 9 to about 5 x 10 1 0 said white blood cells per 150 ml to 200 ml of the starting composition.
7. 9. A process according to any one claims 2 to 8 wherein the number of red blood cells in the starting composition is less than about 3% of the number of total white blood cells. A process according to claim 2 wherein, after washing the starting composition, the number of platelets cultured with the mononuclear cells is reduced by about 80% to about 90% compared to the number of platelets present in the starting composition.
8. 11. A process according to any one of claims 2 to 10 wherein the mononuclear cells ao.. 15 are cultured with other white blood cell said cellular constituents and wherein the monocytes represent about 10% to about 20% of the white blood cells cultured. S12. A process according to claim 11 wherein the white blood cells cultured comprise about 60% to about 80% of lymphocytes.
9. 13. A process according to any one of claims 2 to 12 wherein said starting composition of blood cells is an apheresis product obtained from blood.
10. 14. A process according to any one of claims 1 to 13 wherein the mononuclear cells are cultured in the presence of one of more cytokines and/or adjuvants. 99o 1-00.DOC -27- A process according to claim 14 wherein the one or more cytokines and/or adjuvants are selected from the group consisting of GMCSF, TNF-a, IL-13, IL-4, calcitriol and histamine.
11. 16. A process according to any one of claims 1 to 15 wherein the mononuclear cells are cultured in the presence of an activator of macrophages.
12. 17. A process according to claim 16 wherein the mononuclear cells are cultured with the activator of macrophages for about 16 to about 24 hours.
13. 18. A process according to claim 16 or 17 wherein the activator of macrophages is IFN-y. 10 19. A process according to any one of claims 16 to 18 wherein the mononuclear cells are cultured in the presence of one or more antigens.
14. 20. A process according to any one of claims 1 to 19 wherein the mononuclear cells *0e* are cultured for a period of from about 6 days to about 10 days.
15. 21. A process according to any one of claims 1 to 20 further involving a stage of 15 elutriation following said culturing of the mononuclear cells and which comprises: o causing cultured cells obtained by said culturing to be transfused into an elutriator incorporating an elutriation chamber in which is located a rotor, and having an inlet for entry of the cultured cells into the elutriation chamber and an outlet from said elutriation chamber; (ii) operating the rotor of the elutriator as the cultured cells are transfused into the elutriation chamber through said inlet, whereby the cultured cells are separated in the elutriation chamber on the basis of size; and
16. 1901-00.DOC -28- (iii) collecting different groups of the separated said cultured cells once the groups pass from the outlet of the elutriator. 22. A process according to claim 21 wherein the cultured cells are mixed with an elutriation solution in a transfuser while being transfused into the elutriator, and wherein the transfusing of the cultured cells is effected by a pump which draws said cultured cells and said elutriation solution into the transfuser upon closure of an air intake of the transfuser. 23. A process according to claim 21 or 22 wherein the different groups of the cultured cells are collected into different collection receptacles upon passing from the outlet of 10 the elutriator, respectively. 24. A process according to claim 23 wherein a said group of macrophages or other monocyte derived cells in increased purity relative to that prior to entry into the elutriator is collected. 25. A process according to any one of claims 1 to 24 further comprising the step of 15 removing lymphocytes from other cultured cells present in the culture medium upon completion of said culturing, utilising anti-lymphocyte antibodies. 26. A process according to claim 25 wherein the cultured cells are transferred into one or more receptacles internally coated with the anti-lymphocyte antibodies prior to recovering said cultured cells not bound to the anti-lymphocyte antibodies from the receptacle or receptacles. 27. A process according to any one of claims 1 to 26 wherein the preparation of monocyte derived cells is a preparation ofmacrophages or other monocyte derived cells.
17. 19901-OO.DOC 4 4 4 4 4 4 -29 28. A preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response, or precursors thereof, prepared by a process as defined in any one of claims 1 to 27. 29. A system when used in a method as defined in anyone of claims 1 to 27 for providing a preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response, or precursors thereof wherein the system comprises: at least one first receptacle containing a starting composition of blood cells for being processed; (ii) at least one second receptacle containing a physiologically acceptable 10 solution for washing cellular constituents of the starting composition; (iii) at least one third receptacle for receiving the physiologically acceptable solution and the starting composition of blood cells from the first and second receptacles, respectively; (iv) at least one fourth receptacle containing culture medium; 15 first transfer means for enabling transfer of the physiologically acceptable solution and the starting composition from the first and second receptacles into the third receptacle to dilute the starting composition of blood cells for said washing; and (vi) second transfer means for enabling transfer of the culture medium from the fourth receptacle to cellular constituents of the starting composition washed with the physiologically acceptable solution and which are to be cultured in the culture medium; wherein the starting composition includes suitable mononuclear cells for being cultured and other said cellular constituents including granulocytes. 19')O -00oo DOC A system according to claim 29 further comprising: (vii) at least one culturing receptacle for culturing of the cellular constituents in the culture medium; and wherein said second transfer means enables transfer of the culture medium from the fourth receptacle to the cellular constituents for being cultured, and subsequent transfer of the culture medium and said cellular constituents for being cultured to the culturing receptacle. 31. A system according to claim 30 wherein the second transfer means comprises tubing for the transfer of the culture medium to the cellular constituents for being 10 cultured, and subsequent transfer of the culture medium and said cellular constituents for being cultured, to the culturing receptacle. 32. A system according to claim 30 or 31 wherein the first transfer means comprises tubing for enabling the transfer of the physiologically acceptable solution into the third receptacle and further tubing for enabling the transfer of the starting composition of 15 blood cells into the third receptacle. 33. A system according to claim 32 wherein the tubing for the transfer of the physiologically acceptable solution and the tubing for the transfer of the starting composition of blood cells open into a length of common tubing of the first transfer means, for passage of the physiologically acceptable solution and said starting composition into the third receptacle. 34. A system according to claim 33 wherein the third receptacle is suitable for being centrifuged and the first transfer means is adapted for enabling transfer of supernatant 19901-00 DOC -31 from the third receptacle following centrifugation of the third receptacle to achieve said washing. A system according to claim 34 further comprising at least one supernatant collection receptacle for receiving the supernatant from the first transfer means. 36. A system according to claim 35 wherein the first transfer means incorporates waste supemrnatant tubing for enabling transfer of the supernatant from the third receptacle to the supernatant collection receptacle. 37. A system according to claim 32 further comprising waste supernatant tubing for enabling transfer of supernatant from the third receptacle following centrifugation of the S 10 third receptacle to achieve said washing. 38. A system according to any one of claims 29 to 37 further comprising: at least one further receptacle containing a physiologically acceptable diluent; a transfuser for mixing the diluent with cultured cells when obtained from 15 said culture of the cellular constituents in the culture medium; and third transfer means for enabling transfer of the diluent and the cultured cells to the transfuser. 39. A system according to claim 38 further comprising: an elutriator for receiving the cultured cells in the diluent from the transfuser, and separating the cultured cells into different groups for collection; and fourth transfer means for enabling transfer of the cultured cells in the diluent to the transfuser; 19901-00.DOC -32- wherein the elutriator incorporates an elutriation chamber in which is located a rotor, and has an inlet for entry of the cultured cells into the elutriation chamber, and wherein the rotor of the elutriator is operable when the cultured cells are fed into the elutriation chamber through the inlet to cause the cultured cells to be separated on the basis of size into the said different groups for being collected once said groups have passed from the outlet of the elutriator. A system according to claim 39 further comprising a pump for drawing the diluent and the cultured cells into the transfuser and for feeding the cultured cells in the diluent to the inlet of the elutriation chamber of the elutriator. 10 41. A system according to any one of claims 38 to 40 wherein the transfuser incorporates an air inlet for being closed to enable said transfer of the diluent and the cultured cells to the transfuser. *oo 42. A system according to any one of claims 29 to 41, when used in a process as defined in any one of claims 1 to 27. 0: *0 15 43. A preparation of mononuclear cells capable of presenting antigen to induce a 0- *0 specific immune response, or precursors thereof, prepared with the use of a system as 0 0 0. defined in any one of claims 29 to 41 in a method as defined in any one of claims 1 to 0* 27. 44. A kit when used in a process as defined in any one of claims 1 to 27 wherein the kit comprises at least some components of the system defined in any one of claims 29 to 41. A process for providing a preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response or precursors comprising S1901-00 DOC 4 I -33 culturing mononuclear cells obtained from blood, wherein the mononuclear cells are cultured without prior separation of the mononuclear cells from granulocytes and from red blood cells, substantially as hereinbefore described with reference to one or more of Figures 1 to 3. 46. A system suitable for use in providing a preparation of monocyte derived cells capable of presenting antigen to induce a specific immune response, or precursors thereof, substantialy as hereinbefore described with reference to one or more of Figures 1 to 3. 47. A kit when used in a method for providing a preparation of monocyte derived cells 10 capable of presenting antigen to induce a specific immune response, or precursors thereof, wherein the kit comprises at lease some components of a system substantially as hereinbefore described with reference to one or more of Figures 1 to 3. DATED this 23rd Day of March 2000 Attorney: DAVID ADAMTHWAITE °15 Fellow of the Institute of Patent Attorneys of Australia of BALDWIN SHELSTON WATERS 9* a 19901-00.DOC